The great advances that have occurred in this century in treating infectious diseases are threatened by the emergence of strains of bacteria and other pathogens that are resistant to all currently known antibiotics. To counteract this threat, therapies that have novel mechanisms of action are needed.
One promising target for novel antipathogenic drugs is the topoisomerase family of enzymes. DNA topoisomerases play multiple roles in the maintenance and propagation of the genomes of both prokaryotes and eukaryotes. Thus, compounds that act as effective cellular inhibitors of topoisomerases are expected to act as cytotoxic agents through perturbation of the normal cell division process. Such agents that are sufficiently potent and selective will be of great use as antibacterial and antifungal pharmaceutical agents. Topoisomerases are also encoded by genomes of certain viruses, so development of topoisomerase inhibitors that are effective against viral topoisomerases may provide effective antiviral agents. In addition, because cell division is an important characteristic of cancers and other proliferative diseases, agents that inhibit topoisomerases will also find use as antineoplastic agents.
Topoisomerase inhibitors are classified into two general types. First, the class designated as "poisons" have in common the property of causing "trapping" of the target topoisomerase in the form of a covalent complex with the nucleic acid substrate. Second, the "non-poison" class inhibits the enzymatic activity of the topoisomerase without specific effects on steps of the catalytic cycle that involve formation or resolution of the enzyme-DNA covalent intermediate. Of the DNA topoisomerase inhibitors currently used as clinical antibiotic or antineoplastic agents, the "poisons" seem to be most effective, probably because such compounds result in the accumulation of irreversible genotoxic damage in target cells.
Shortcomings with previously available assays for topoisomerasc inhibitors have hampered the search for novel topoisomerase inhibitors. For example, many previously available assays require the use of radioactive compounds and/or suffer from a lack of sensitivity. Also, previously available topoisomerase inhibitor assays are not always amenable to high throughput screening methods such as are needed to screen large libraries or groups of potential inhibitors. Thus, a need exists for new assay methods for identifying topoisomerase inhibitors. The present invention meets this and other needs.